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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.

Natural 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]

Background: Streptomyces roseochromogenes NRRL 3504 produces clorobiocin, an aminocoumarin antibiotic that inhibits DNA replication. No other natural products have been isolated from this bacterium so far, despite the presence of a rich repertoire of specialized metabolite biosynthesis gene clusters (smBGCs) within its genome. Heterologous expression of smBGCs in suitable chassis speeds up the discovery of the natural products hidden behind these sets of genes. Results: In this work we focus on one intriguing smBGC of NRRL 3504 bearing some similarity to gene clusters involved in production of manumycin family polyketides. Through heterologous expression in Streptomyces chassis strains S. albus Del14 and S. lividans ΔYA9, this smBGC (hereafter referred to as lim BGC) was shown to direct the production of unusual polyketide limocrocin (LIM) known for its ability to interfere with viral reverse transcriptases. The organization of lim BGC, data on the structures of revealed metabolites as well as manipulations of lim genes allowed us to put forward an initial hypothesis about a biosynthetic pathway leading to LIM. We provide initial data on two LIM derivatives as well as updated NMR spectra for the main product. Conclusion: This study reveals the genetic control of biosynthesis of LIM that remained hidden for the last 70 years. This, in turn, opens the door to biological routes towards overproduction of LIM as well as generation of its derivatives. [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.)

Background: The biosynthesis of the natural product family of the polycyclic tetramate macrolactams (PoTeMs) employs an uncommon iterative polyketide synthase/non-ribosomal peptide synthetase (iPKS/NRPS). This machinery produces a universal PoTeM biosynthetic precursor that contains a tetramic acid moiety connected to two unsaturated polyene side chains. The enormous structural and hence functional diversity of PoTeMs is enabled by pathway-specific tailoring enzymes, particularly cyclization-catalyzing oxidases that process the polyene chains to form distinct ring systems, and further modifying enzymes. Results: Ikarugamycin is the first discovered PoTeM and is formed by the three enzymes IkaABC. Utilizing the iPKS/NRPS IkaA, we established a genetic plug-and-play system by screening eight different strong promoters downstream of ikaA to facilitate high-level heterologous expression of PoTeMs in different Streptomyces host systems. Furthermore, we applied the system on three different PoTeM modifying genes (ptmD, ikaD, and cftA), showing the general utility of this approach to study PoTeM post-PKS/NRPS processing of diverse tailoring enzymes. Conclusion: By employing our plug-and-play system for PoTeMs, we reconstructed the ikarugamycin biosynthesis and generated five derivatives of ikarugamycin. This platform will generally facilitate the investigation of new PoTeM biosynthetic cyclization and tailoring reactions in the future. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved. [ABSTRACT FROM AUTHOR]

Enzymatic depolymerisation is increasingly recognised as a reliable and environmentally friendly method. The development of this technology hinges on the availability of high-quality enzymes and associated bioreaction systems for upscaling biodegradation. Microbial heterologous expression systems have been studied for meeting this demand. Among these systems, the Pichia pastoris expression system has emerged as a widely used platform for producing secreted heterologous proteins. This article provides an overview of studies involving the recombinant expression of polymer-degrading enzymes using the P. pastoris expression system. Research on P. pastoris expression of interested enzymes with depolymerising ability, including cutinase, lipase, and laccase, are highlighted in the review. The key factors influencing the heterologous expression of polymer-degrading enzymes in P. pastoris are discussed, shedding light on the challenges and opportunities in the development of depolymerising biocatalysts through the P. pastoris expression system. [ABSTRACT FROM AUTHOR]

In this study, the codon bias analysis of FcF6′H1 gene in Fraxinus chinensis was analyzed using CodonW software and EMBOSS online program, cluster analysis, neutral plot, ENC-plot and PR2-plot of the codon of FcF6′H1 gene in Rutaceae, Leguminosae, Umbelliferae, Oleaceae, Arabidopsis, tobacco and tomato was conduceted, the factors affecting information of the codon bias of the FcF6′H1 gene in Fraxinus chinensis were studied, the best receptors were obtained by comparing the codon usage frequencies of FcF6′H1 and model organisms. The results showed that the content of GC、GC1、 GC2、GC3, and GC12 of FcF6′H1 in Fraxinus chinensis was 0.431 2, 0.512 5, 0.349 0, 0.432 1, and 0.430 8, respectively, CAI was 0.216, and ENC was 57.84, indicating weak codon bias of FcF6′H1 in Fraxinus chinensis. The results of neutral analysis, ENC-plot analysis, and PR2-plot analysis indicated that codon bias of FcF6′H1 was affected by base mutation and natural selection. The results of CDS evolutionary tree and RSCU cluster analysis were not completely consistent, but both indicated that FcF6′H1 in Fraxinus chinensis and XM_023028611.1, XM_023036788.1, and XM_023034893.1 of Olea europaea var. sylvestris were aggregated into one category. Six optimal codons of F6′H1 gene were identified, they were CUC, AUC, AAG, GAG、UCG, and ACA. The codon usage frequence analysis showed that in the model plants, tomato tobacco were suitable for the genetic transformation receptors, and the Escherichia coli expression system was suitable for the heterologous expression vector of FcF6′H1. This study preliminarily clarified the codon usage pattern of FcF6′H1 gene in Fraxinus chinensis. [ABSTRACT FROM AUTHOR]

Key message: A total of 24 genes of vacuolar H+-translocating pyrophosphatases H+-PPases (VPP) genes were identified in Saccharum spontaneum AP85-441 and the ScVPP1-overexpressed Arabidopsis plants conferred salt tolerance. The vital role of vacuolar H+-translocating pyrophosphatases H+-PPases (VPP) genes involved in plants in response to abiotic stresses. However, the understanding of VPP functions in sugarcane remained unclear. In this study, a total of 24 VPP genes (SsaVPP1–SsaVPP24) were identified in the Saccharum spontaneum genome of haploid clone AP85-441. These genes were distributed in two phylogenetic groups. The SsaVPPs displayed diverse physio-chemical and gene structure attributes. The SsaVPP family genes have expanded through segmental duplication (20 gene pairs) rather than tandem duplication. A full-length cDNA of ScVPP1 was cloned from the sugarcane cultivar ROC22 and shared 99.48% sequence identity (amino acid) with homologous gene SsaVPP21 from AP85-441. In ROC22, the ScVPP1 gene was considerably upregulated by NaCl and ABA treatments among leaf, root, and stem tissues, while this gene was exclusively upregulated in the root with PEG treatment. Under NaCl and ABA stresses, yeast cells transfected by the ScVPP1 plasmid showed distinct growth rates compared to control yeast cells transfected by the empty vector. In transgenic Arabidopsis lines overexpressing ScVPP1, the seed gemination and survival rate were enhanced under NaCl treatment but not under ABA stress as compared to wild-type plants. These results suggested that the ScVPP1 gene conferred tolerance to slat and may be used as a salt resistance gene source for sugarcane breeding. [ABSTRACT FROM AUTHOR]

Copyright of Journal of South-Central Minzu University (Natural Science Edition) is the property of Journal of South-Central Minzu University (Natural Science Edition) 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.)

Biosynthesis of sodorifen with a unique C 16 -bicyclo[3.2.1]octene framework requires an S -adenosyl methionine-dependent methyltransferase SodC and terpene cyclase SodD. While bioinformatic analyses reveal a wide distribution of the sodCD genes organization in bacteria, their functional diversity remains largely unknown. Herein, two sodorifen-type gene clusters, pcch and pcau , from Pseudomonas sp. are heterologously expressed in Escherichia coli , leading to the discovery of two C 16 terpenoids. Enzymatic synthesis of these compounds is achieved using the two (SodCD-like) pathway-specific enzymes. Enzyme assays using different combinations of methyltransferases and terpene synthases across the pcch , pcau , and sod pathways reveal a unifying biosynthetic mechanism: all three SodC-like enzymes methylate farnesyl pyrophosphate (FPP) with subsequent cyclization to a common intermediate, pre-sodorifen pyrophosphate. Structural diversification of this joint precursor solely occurs by the subsequently acting individual terpene synthases. Our findings expand basic biosynthetic understanding and structural diversity of unusual C 16 -terpenoids. [Display omitted] • Two non-canonical volatile C 16 terpenoids from Pseudomonas sp. were identified • The biosyntheses of pseudorifen and aristotelene were reconstituted in vitro • Product diversification from a joint precursor is achieved by terpene synthases Mo et al. identified the gene clusters directing biosynthesis of non-canonical C 16 terpenoids from Pseudomonas species and reconstituted their biosynthetic pathways in vitro , revealing that biosynthesis of these C 16 terpenoids is performed via a uniform intermediate, with product diversification solely achieved by the terpene synthases. [ABSTRACT FROM AUTHOR]

Background: Gaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. Here, we provide evidence demonstrating that two highly selected mutations in CYP6P4a and CYP6P4b are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa. Results: Continent-wide polymorphism survey revealed escalated signatures of directional selection of both genes between 2014 and 2021. In vitro insecticide metabolism assays with recombinant enzymes from both genes showed that mutant alleles under selection exhibit higher metabolic efficiency than their wild-type counterparts. Using the GAL4-UAS expression system, transgenic Drosophila flies overexpressing mutant alleles exhibited increased resistance to pyrethroids. These findings were consistent with in silico predictions which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we designed two DNA-based assays for the detection of CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce killing effects of pyrethroid bed nets. Conclusions: Overall, this study demonstrated that CYP6P4a and CYP6P4b contribute to pyrethroid resistance in An. funestus and provided two additional insecticide resistance molecular diagnostic tools that would contribute to monitoring and better management of resistance. [ABSTRACT FROM AUTHOR]

The exploration and exploitation of deep-sea microbial resources is of great scientific value for understanding biological evolution under extreme conditions. Deep-sea microorganisms are critical in the ocean carbon cycle, and marine heterotrophic microorganisms secrete extracellular carbonic anhydrase (CA) to fix inorganic carbon, an important process in climate regulation. Extracellular CA provides a green method for fixing carbon dioxide into stable minerals containing Ca2+. However, studies on extracellular CA in deep-sea microorganisms are limited. In this study, Bacillus safensis yw6 was isolated from Mariana Trench sediments and three candidate extracellular CA genes (β-ca1, β-ca2, and γ-ca) were identified by whole genome sequencing. Bioinformatics analyses showed that these CAs have different structural compositions, with the β-CA having α-helix and random coiling, whereas the γ-CA has more random coiling and stretched strands. Heterologous expression in E. coli BL21 (DE3) showed that β-CA2 had the highest enzyme activity, followed by γ-CA and β-CA1. Field emission scanning electron microscopy (FESEM) observations showed that the engineered strains with β-ca2 genes produced deposits that were like those from natural sources. This finding not only provides new perspectives for the utilization of deep-sea microbial resources, but also provides an important scientific basis for the molecular mechanisms of extracellular CAs of deep-sea microbes. [ABSTRACT FROM AUTHOR]

The objective of the study: Abiotic stress significantly threatens plant survival. In response to challenging conditions, plants have evolved various resistance mechanisms. Recently, genetic strategies have become increasingly prevalent in efforts to enhance abiotic stress tolerance and develop stress-resistant plant varieties. In this study, we employed heterologous expression of two metabolism-related haloarchaeal genes in the Nicotiana tabacum genome to improve its salt and drought tolerance mechanisms. Experimental procedure(s): The U2642 and CL116 genes encoding 3-hydroxybutyryl-CoA dehydrogenase and UDP-glucose 4-epimerase, respectively, were subcloned into the pIPKb004 vector under the control of Cauliflower mosaic virus 35S (CaMV35S) promoter using the Gateway cloning system. The recombinant vectors were introduced into the N. tabacum through an Agrobacterium-mediated gene transfer procedure. Both wild-type (WT) and transgenic plants were cultivated under conditions of 175 mM NaCl and 200 mM mannitol, alongside a control group grown without stress. The transgenic plants were evaluated based on morphological characteristics, germination rates, gene expression levels, proline and malondialdehyde (MDA) accumulation, and antioxidant enzyme activities. Key results: The transgenic lines exhibited enhanced tolerance to abiotic stress, particularly drought, as demonstrated by higher germination rates, increased proline accumulation, reduced MDA production, and elevated antioxidant enzyme activities compared to WT plants. Conclusions: The metabolism-related genes from haloarchaea conferred significant resistance to abiotic stress when heterologously expressed in tobacco, highlighting their potential as promising candidates for developing stress-tolerant crops. [ABSTRACT FROM AUTHOR]

Climate change and human activities impose severe biotic and abiotic stresses that threaten plant survival. To cope with these extreme conditions, plants have evolved various resistance mechanisms. In recent years, genetic approaches have been increasingly employed to enhance abiotic stress tolerance and develop stress-resistant plants. In this study, we introduced three transporter-related genes from extreme haloarchaea, which thrive in saturated NaCl environments, into the Nicotiana tabacum genome to improve its salt and drought tolerance mechanisms. The U2845, U3508, and CL165 genes were subcloned into the pIPKb004 vector, which includes the cauliflower mosaic virus 35S (CaMV35S) promoter and a hygromycin phosphotransferase II (hptlI) selection marker, using Gateway cloning. The recombinant vector was introduced into the N. tabacum genome via Agrobacterium-mediated transformation. Wild-type and transgenic plants were cultivated under 150 mM NaCl and 200 mM mannitol, along with a no-stress control. The effects of these genes were evaluated through morphological examination, germination rate analysis, proline and malondialdehyde accumulation measurements, and antioxidant enzyme activity assays. The transgenic lines demonstrated enhanced stress tolerance, as evidenced by improved germination rates, increased proline accumulation, and elevated antioxidant enzyme activities. These findings underscore the potential of haloarchaeal genes as valuable genetic resources for developing stress-tolerant plants. [ABSTRACT FROM AUTHOR]

Flammulina filiformis is one of the widely produced edible fungi worldwide. It is rich in γ-aminobutyric acid (GABA), a non-protein amino acid with important physiological functions in humans. To investigate the functions of key genes in the GABA metabolic pathway of F. filiformis, we isolated the monokaryon Fv-HL23-1 from the factory-cultivated F. filiformis strain Fv-HL23 and then sequenced and assembled the genome using the PacBio Sequel and Illumina NovaSeq sequencing platforms. The results showed that the genome comprised 140 scaffolds with a total length of 40.96 Mb, a GC content of 49.62%, an N50 of 917,125 bp, and 14,256 protein-coding genes. Phylogenetic analysis based on the whole genome revealed a close evolutionary relationship of Fv-HL23-1 with Armillaria mellea, Lentinula edodes, and Schizophyllum commune. A total of 589 carbohydrate-active enzymes were identified in the genome of Fv-HL23-1, suggesting its strong lignocellulose degradation ability, and 108 CYP450 gene family members were identified, suggesting important functions such as resistance to stress, secondary metabolite synthesis, and growth and development. The F. filiformis proteins glutamate decarboxylase 1 (Ff-GAD1) and glutamate decarboxylase 2 (Ff-GAD2), which may be responsible for GABA synthesis, were identified by protein alignment. Molecular docking analysis showed that Ff-GAD2 may have better catalytic activity than Ff-GAD1. To verify the function of Ff-gad2, its heterologous expression in the mycelia of the mononuclear Hypsizigus marmoreus was analyzed. Compared with wild type, the GABA content of mycelia was increased by 85.40–283.90%, the growth rate was increased by 9.39 ± 2.35%, and the fresh weight was increased by 18.44 ± 7.57%. Ff-GAD2 may play a catalytic role in GABA synthesis. In addition, the expression of the full-length Ff-gad2 gene was increased by 7.96 ± 1.39 times compared with the exon expression level in H. marmoreus mycelia, suggesting that the intron may contribute to the heterologous expression of Ff-GAD2. Based on whole-genome sequencing, we analyzed the enzyme system related to the important life activities of F. filiformis, focusing on the function of Ff-GAD, a key enzyme in the GABA synthesis pathway. The results lay a foundation for elucidating the GABA metabolism pathway of edible fungi and developing targeted breeding strategies for GABA-producing edible fungi. [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. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Aims The main objective of this study was to produce erythronolide B (EB) and 3- O- α-mycarosylerythronolide B (MEB) in Streptomyces coelicolor and enhance the MEB production by expressing the glucose-1-phosphate thymidylyltransferase (RfbA). Methods and results We expressed eryF and eryB genes (eryBII, eryBIII, eryBIV, eryBV, eryBVI , and eryBVII) to produce EB and MEB. The expression was confirmed by quantitative real-time polymerase chain reaction. Furthermore, the MEB's production was improved by more than 100-fold by expressing an enzyme, RfbA, which is absent from the erythromycin gene cluster, to promote the biosynthesis of TDP-L-mycarose. We discuss the feasibility of alternative Streptomyces species for erythromycin production based on the presence or absence of RfbA. Conclusions The RbfA enzyme from Saccharopolyspora erythraea was expressed in S. coelicolor M1152 along with the MEB biosynthesis pathway, resulting in a large increase in MEB production (>100-fold). [ABSTRACT FROM AUTHOR]

The increasing prevalence of drug-resistant bacteria has prompted the search for alternative approaches to address bacterial infections. Lanthipeptides, which are small molecule peptides, are biosynthesized and post-translationally modified by ribosomes in gram-positive bacteria. These compounds possess distinctive target recognition properties, making them highly potential candidates for combating antimicrobial resistance. Consequently, they hold promising potential as innovative substitutes for traditional antibiotics. We conducted a comprehensive analysis of 65 publicly available Bacillus amyloliquefaciens genomes' data to explore the potential for lanthipeptide synthesis in this species. We annotated 21 putative precursor peptide genes for these novel lanthipeptides. Through heterologous expression experiments, we successfully obtained a novel lanthipeptide named amyA3. We accomplished a novel co-transformation strategy by introducing the synthetic enzyme obtained from B. amyloliquefaciens WS-8 into the E. coli BL21 (DE3) strain along with the heterologous precursor peptide amyA3. Through mass spectrometry analysis, we confirmed the successful modification of amyA3 by the synthetic enzyme derived from B. amyloliquefaciens WS-8, which represents a significant breakthrough in this research field. The identification and characterization of the lanthipeptide amyA3 along with the elucidation of its biosynthetic pathway provide novel perspectives into the discovery of naturally derived products and the intricate processes of translation and post-translational modifications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Transcription activation by positive regulators and their regulating sequences. • Positive regulator stimulates transcription of itself and downstream genes. • Higher transcription activation than strong constitutive promoters. • Demonstrated in Pseudomonas stutzeri , Escherichia coli and Streptomyces albus. • Highest yields of indole-3-acetic acid and prodigiosin in heterologous hosts. Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. Development of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. We designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. When this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. Many positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology. [ABSTRACT FROM AUTHOR]

Hericium erinaceus (Lion's Mane mushroom) is widely consumed for its numerous reported benefits for brain health. A growing body of evidence suggests that these benefits are likely attributable to aromatic compounds contained in its fruiting bodies, including the meroterpenoids hericenones. Here, we report the identification and reconstitution of the first two steps of the biosynthetic pathway of hericenones via heterologous expression of the polyketide synthase HerA and the carboxylic acid reductase HerB in Aspergillus oryzae. Furthermore, we investigated a putative prenyltransferase that might be responsible for the following biosynthetic step. Ongoing efforts to reconstitute the full pathway will enable large-scale production of hericenones and other meroterpenoids in heterologous hosts. [ABSTRACT FROM AUTHOR]

The escalating threat of ultraviolet radiation (UVR) necessitates the development of sustainable strategies for protection from UVR exposure. Mycosporine-like amino acids (MAAs) provide natural photoprotection by absorbing and dissipating harmful UVR. Here, we present the engineering of Nannochloropsis gaditana, a marine microalga, for enhanced production of MAAs, specifically shinorine. A novel strain of N. gaditana isolated from Seonjaedo Island, Korea, served as a heterologous host for MAA production. By introducing MAA biosynthetic genes from Pyropia yezoensis and Nostoc punctiforme into the N. gaditana host strain, we generated two transgenic N. gaditana strains capable of overproducing shinorine. The transgenic N. gaditana strains exhibited enhanced resistance to UVR-induced damage, as evidenced by greater photosystem II efficiency and lower reactive oxygen species generation upon exposure to UVR compared with the unmodified host strain. We also discuss the substrate specificity of key enzymes involved in MAA biosynthesis in the transgenic N. gaditana strains. Our study underscores the feasibility of utilizing microalgae for sustainable MAA production and provides insights into the optimization of MAA biosynthesis pathways for commercialization and environmental sustainability. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development 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.)

In this study, the probiotic yeast Saccharomyces boulardii was engineered to secrete the antiviral lectin griffithsin. Twelve genetic tools with the griffithsin gene were cloned into the vector pSF-TEF1-URA3 and introduced into S. boulardii. In the recombinant strains, a 16.9 kDa band was detected using SDS-PAGE and further recognized by griffithsin antibody with Western blotting. S. boulardii strains FM, FT, HC, and HE with a high yield of griffithsin were acquired for property characterization in vitro. The four recombinant strains displayed a similar growth pattern to that of the control strains, while their morphological characteristics had changed according to scanning electron microscopy. In simulated gastrointestinal digestive fluids, the survival rates of S. boulardii FM, FT, and HC were significantly decreased (86.32 ± 1.49% to 95.36 ± 1.94%) compared with those of the control strains, with survival rates between 95.88 ± 0.00% and 98.74 ± 1.97%. The hydrophobicity of S. boulardii FM, the strain with the highest griffithsin production, was significantly increased to 21.89 ± 1.07%, and it exhibited a reduced auto-aggregation rate (57.64 ± 2.61%). Finally, Vero cells infected with porcine epidemic diarrhea virus (PEDV) were used to evaluate the strains' antiviral activity, and the rate at which S. boulardii FM inhibited PEDV reached 131.36 ± 1.06%, which was significantly higher than that of the control group. [ABSTRACT FROM AUTHOR]

Abstract Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved. Graphical Abstract

Background. Methionine aminopeptidases (MAPs) are a class of enzymes that catalyze the removal of the N-terminal initiator methionine from a polypeptide chain. Bacterial MAPs are considered as targets for the development of broad-spectrum antibacterial drugs, and using MAPs in biotechnology necessitates the search for new MAPs and the study of their functioning and inhibition mechanisms.The aim of the study. To identify methionine aminopeptidase in the Thermus thermophilus genome (Tt-MAP) and to confirm its functional activity.Materials and methods. To identify Tt-MAP, we analyzed the Thermus thermophilus genome in the GeneBank database. Modern genetic engineering techniques (polymerase chain reaction, restriction, transformation, heterologous expression) were used to clone the putative open reading frame (ORF) encoding Tt-MAP in the pHUE vector. Various chromatography techniques (affinity, ion exchange, and size-exclusion) were used to obtain a purified enzyme preparation. The fluorogenic substrate L-methionine 7-amino-4-methylcoumarin (Met-AMC) was used to confirm the specific functional aminopeptidase activity of the enzyme.Results. An ORF encoding MAP was identified in the Thermus thermophilus bacterium genome. Oligonucleotide primers were designed based on the nucleotide sequence. The ORF was cloned in the vector, and the recombinant enzyme was produced in E. coli cells. A method for purifying the enzyme to a homogeneous state was developed using a series of sequential chromatographies, allowing up to 30 mg to be obtained from 1 liter of culture. Using the fluorogenic substrate Met-AMC, the specific functional activity of the enzyme was demonstrated (the enzyme cleaves methionine from the substrate).Conclusion. We have identified the Thermus thermophilus MAP and tested its functional activity. It has been shown that the ORF product TTHA1670 encodes a methionine-specific aminopeptidase, i. e. methionine aminopeptidase. The enzyme can be used in various fields of biotechnology and scientific research.

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.

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

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.