Discovery and Heterologous Production of New Cyclic Depsibosamycins.
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ABSTRACT:
SUBMITTER: Stierhof M
PROVIDER: S-EPMC8303602 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.
| S-EPMC5900794 | biostudies-literature
Project description:The intriguing structural complexity of molecules produced by natural organisms is uncontested. Natural scaffolds serve as an important basis for the development of molecules with broad applications, e.g., therapeutics or agrochemicals. Research in recent decades has demonstrated that by means of classic metabolite extraction from microbes only a small portion of natural products can be accessed. The use of genome mining and heterologous expression approaches represents a promising way to discover new natural compounds. In this paper we report the discovery of a novel cyclic pentapeptide called bonsecamin through the heterologous expression of a cryptic NRPS gene cluster from Streptomyces albus ssp. chlorinus NRRL B-24108 in Streptomyces albus Del14. The new compound was successfully isolated and structurally characterized using NMR. The minimal set of genes required for bonsecamin production was determined through bioinformatic analysis and gene deletion experiments. A biosynthetic route leading to the production of bonsecamin is proposed in this paper.
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Project description:Cyanobacteria are oxygenic photosynthetic prokaryotes that play important roles in the global carbon cycle. Recently, engineered cyanobacteria capable of producing various small molecules from CO2 have been developed. However, cyanobacteria are seldom considered as factories for producing proteins, mainly because of the lack of efficient strong promoters. Here, we report the discovery and verification of a super-strong promoter P(cpc560), which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Using P(cpc560), functional proteins were produced at a level of up to 15% of total soluble protein in the cyanobacterium Synechocystis sp. 6803, a level comparable to that produced in Escherichia coli. We demonstrated that the presence of multiple TFBSs in P(cpc560) is crucial for its promoter strength. Genetically transformable cyanobacteria neither have endotoxins nor form inclusion bodies; therefore, P(cpc560) opens the possibility to use cyanobacteria as alternative hosts for producing heterogeneous proteins from CO2 and inorganic nutrients.
| S-EPMC3968457 | biostudies-literature
Project description:BackgroundCyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce.ResultsWe isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production.ConclusionsIn this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.
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Project description:SummaryCurcuminoids, components of the rhizome of turmeric, show several beneficial biological activities, including anticarcinogenic, antioxidant, anti-inflammatory, and antitumor activities. Despite their numerous pharmaceutically important properties, the low natural abundance of curcuminoids represents a major drawback for their use as therapeutic agents. Therefore, they represent attractive targets for heterologous production and metabolic engineering. The understanding of biosynthesis of curcuminoids in turmeric made remarkable advances in the last decade, and as a result, several efforts to produce them in heterologous organisms have been reported. The artificial biosynthetic pathway (e.g., in Escherichia coli) can start with the supplementation of the amino acid tyrosine or phenylalanine or of carboxylic acids and lead to the production of several natural curcuminoids. Unnatural carboxylic acids can also be supplemented as precursors and lead to the production of unnatural compounds with possibly novel therapeutic properties. In this paper, we review the natural conversion of curcuminoids in turmeric and their production by E. coli using an artificial biosynthetic pathway. We also explore the potential of other enzymes discovered recently or already used in other similar biosynthetic pathways, such as flavonoids and stilbenoids, to increase curcuminoid yield and activity.
| S-EPMC4402967 | biostudies-literature
Project description:Hyaluronic Acid (HA) is a biopolymer composed by the monomers Glucuronic Acid (GlcUA) and N-Acetyl Glucosamine (GlcNAc). It has a broad range of applications in the field of medicine, being marketed between USD 1000-5000/kg. Its primary sources include extraction of animal tissue and fermentation using pathogenic bacteria. However, in both cases, extensive purification protocols are required to prevent toxin contamination. In this study, aiming at creating a safe HA producing microorganism, the generally regarded as safe (GRAS) yeast Kluyveroymyces lactis is utilized. Initially, the hasB (UDP-Glucose dehydrogenase) gene from Xenopus laevis (xlhasB) is inserted. After that, four strains are constructed harboring different hasA (HA Synthase) genes, three of humans (hshasA1, hshasA2, and hshasA3) and one with the bacteria Pasteurella multocida (pmhasA). Transcript values analysis confirms the presence of hasA genes only in three strains. HA production is verified by scanning electron microscopy in the strain containing the pmHAS isoform. The pmHAS strain is grown in a 1.3 l bioreactor operating in a batch mode, the maximum HA levels are 1.89 g/L with a molecular weight of 2.097 MDa. This is the first study that reports HA production in K. lactis and it has the highest HA titers reported among yeast.
| S-EPMC6780701 | biostudies-literature
Project description:BackgroundSerratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase.MethodsThe heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein.ResultsSerratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37° C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction.ConclusionAs serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.
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Project description:Modified starches offer a biodegradable, readily available, and cost-effective alternative to petroleum-based products. The reaction of alkenylsuccinic anhydrides (ASAs), in particular, is an efficient method to produce amphiphilic starches with numerous applications in different areas. While ASAs are typically derived from petroleum sources, maleated soybean oil can also be used in an effort to produce materials from renewable sources. The reaction of gelatinized waxy maize starch with octenylsuccinic anhydride (OSA), dodecenylsuccinic anhydride (DDSA), a maleated fatty acid (TENAX 2010), phthalic anhydride (PA), 1,2,4-benzenetricarboxylic acid anhydride (trimellitic anhydride, TMA), and three maleated soybean oil samples, was investigated under different conditions. To minimize the reaction time and the amount of water required, the outcome of the esterification reaction was compared for starch dispersions in benchtop dispersed reactions, for starch melts in a heated torque rheometer, and for reactive extrusion in a pilot plant scale twin-screw extruder. The extent of reaction was quantified by 1H NMR analysis, and changes in molecular weight and diameter were monitored by gel permeation chromatography (GPC) analysis. The outcome of the reactions varied markedly in terms of reaction efficiency (RE), molecular weight distribution, and average hydrodynamic diameter, for the products derived from the different maleated reagents used, as well as for the different reaction protocols.
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Project description:Filamentous fungi are important factories in the production and secretion of homologous and heterologous lignocellulolytic enzymes, however the regulation of protein secretion in these organisms need to be further explored. In order to investigate this regulation, Aspergillus nidulans recombinant strains were analyzed by transcriptomics. We designed three A. nidulans recombinant strains producing the following heterologous proteins: alpha-arabinofuranosidase (AbfA), beta-glucosidase (BglC) and thermophilic mannanase (1542). The heterologous genes abfA and bglC were highly expressed, while the levels of 1542 mRNA were similar to the reference gene. An indirect relationship between mRNA and the levels of protein secretion was observed, suggesting that transcription is not a bottleneck in these systems. Based on the general analysis of RNA-seq of the recombinant strains, it was possible to observe a predominant up-regulation response. Moreover, biological processes related to metabolism, protein with binding function and cellular transport were overrepresented. We also observed the unconventional splicing of hacA for the recombinant A. nidulansAbfA and A. nidulans1542, indicating some level of unfolded protein response. The global analysis showed mild stress at 2 h induction of heterologous protein production, which was normalized after 8 h. Our results provide insights to understand how A. nidulans adapts to the overproduction of heterologous proteins.
2020-07-14 | GSE101522 | GEO
Project description:Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.
| S-EPMC1317351 | biostudies-literature