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Biology May 2024Crosstalk regulation is widespread in species. Elucidating the influence of a specific regulator on target biosynthetic gene clusters (BGCs) and cell metabolism is...
Crosstalk regulation is widespread in species. Elucidating the influence of a specific regulator on target biosynthetic gene clusters (BGCs) and cell metabolism is crucial for strain improvement through regulatory protein engineering. PteF and PteR are two regulators that control the biosynthesis of filipin, which competes for building blocks with avermectins in . However, little is known about the effects of PteF and PteR on avermectin biosynthesis. In this study, we investigated their impact on avermectin biosynthesis and global cell metabolism. The deletion of resulted in a 55.49% avermectin titer improvement, which was 23.08% higher than that observed from deletion, suggesting that PteF plays a more significant role in regulating avermectin biosynthesis, while PteF hardly influences the transcription level of genes in avermectin and other polyketide BGCs. Transcriptome data revealed that PteF exhibited a global regulatory effect. Avermectin production enhancement could be attributed to the repression of the tricarboxylic acid cycle and fatty acid biosynthetic pathway, as well as the enhancement of pathways supplying acyl-CoA precursors. These findings provide new insights into the role of PteF on avermectin biosynthesis and cell metabolism, offering important clues for designing and building efficient metabolic pathways to develop high-yield avermectin-producing strains.
PubMed: 38785828
DOI: 10.3390/biology13050344 -
Frontiers in Microbiology 2024The cultivation system requires that the approach providing biomass for all types of metabolic analysis is of excellent quality and reliability. This study was conducted...
The cultivation system requires that the approach providing biomass for all types of metabolic analysis is of excellent quality and reliability. This study was conducted to enhance the efficiency and yield of antifungal substance (AFS) production in 3-10 by optimizing operation conditions of aeration, agitation, carbon source, and incubation time in a fermenter. Dissolved oxygen (DO) and pH were found to play significant roles in AFS production. The optimum pH for the production of AFS in 3-10 was found to be 6.5. As the AFS synthesis is generally thought to be an aerobic process, DO plays a significant role. The synthesis of bioactive compounds can vary depending on how DO affects growth rate. This study validates that the high growth rate and antifungal activity required a minimum DO concentration of approximately 20% saturation. The DO supply in a fermenter can be raised once agitation and aeration have been adjusted. Consequently, DO can stimulate the development of bacteria and enzyme production. A large shearing effect could result from the extreme agitation, harming the cell and deactivating its products. The highest inhibition zone diameter (IZD) was obtained with 3% starch, making starch a more efficient carbon source than glucose. Temperature is another important factor affecting AFS production. The needed fermentation time would increase and AFS production would be reduced by the too-low operating temperature. Furthermore, large-scale fermenters are challenging to manage at temperatures that are far below from room temperature. According to this research, 28°C is the ideal temperature for the fermentation of 3-10. The current study deals with the optimization of submerged batch fermentation involving the modification of operation conditions to effectively enhance the efficiency and yield of AFS production in 3-10.
PubMed: 38784807
DOI: 10.3389/fmicb.2024.1378834 -
MBio Jun 2024Bacteriophages, viruses that specifically target plant pathogenic bacteria, have emerged as a promising alternative to traditional agrochemicals. However, it remains...
UNLABELLED
Bacteriophages, viruses that specifically target plant pathogenic bacteria, have emerged as a promising alternative to traditional agrochemicals. However, it remains unclear how phages should be applied to achieve efficient pathogen biocontrol and to what extent their efficacy is shaped by indirect interactions with the resident microbiota. Here, we tested if the phage biocontrol efficacy of phytopathogenic bacterium can be improved by increasing the phage cocktail application frequency and if the phage efficacy is affected by pathogen-suppressing bacteria already present in the rhizosphere. We find that increasing phage application frequency improves density control, leading to a clear reduction in bacterial wilt disease in both greenhouse and field experiments with tomato. The high phage application frequency also increased the diversity of resident rhizosphere microbiota and enriched several bacterial taxa that were associated with the reduction in pathogen densities. Interestingly, these taxa often belonged to known for antibiotics production and soil suppressiveness. To test if they could have had secondary effects on biocontrol, we isolated Actinobacteria from and genera and tested their suppressiveness to the pathogen and . We found that these taxa could clearly inhibit growth and constrain bacterial wilt disease, especially when combined with the phage cocktail. Together, our findings unravel an undiscovered benefit of phage therapy, where phages trigger a second line of defense by the pathogen-suppressing bacteria that already exist in resident microbial communities.
IMPORTANCE
is a highly destructive plant-pathogenic bacterium with the ability to cause bacterial wilt in several crucial crop plants. Given the limitations of conventional chemical control methods, the use of bacterial viruses (phages) has been explored as an alternative biological control strategy. In this study, we show that increasing the phage application frequency can improve the density control of , leading to a significant reduction in bacterial wilt disease. Furthermore, we found that repeated phage application increased the diversity of rhizosphere microbiota and specifically enriched Actinobacterial taxa that showed synergistic pathogen suppression when combined with phages due to resource and interference competition. Together, our study unravels an undiscovered benefit of phages, where phages trigger a second line of defense by the pathogen-suppressing bacteria present in resident microbial communities. Phage therapies could, hence, potentially be tailored according to host microbiota composition to unlock the pre-existing benefits provided by resident microbiota.
Topics: Rhizosphere; Ralstonia solanacearum; Microbiota; Solanum lycopersicum; Plant Diseases; Soil Microbiology; Bacteriophages; Actinobacteria
PubMed: 38780276
DOI: 10.1128/mbio.03016-23 -
Synthetic and Systems Biotechnology Sep 2024offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and...
offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and increased yield of specific compounds in have been technically challenging due to their slow growth rate, complex culture conditions and intricate genetic backgrounds. In this study, we screened dozens of strains inhabiting in a plant rhizosphere for fast-growing candidates, and further employed CRISPR/Cas-based engineering techniques for stepwise refinement of a particular strain, sp. A-14 that harbors a 7.47 Mb genome. After strategic removal of nonessential genomic regions and most gene clusters, we reduced its genome size to 6.13 Mb, while preserving its growth rate to the greatest extent. We further demonstrated that cleaner metabolic background of this engineered strain was well suited for the expression and characterization of heterologous gene clusters, including the biosynthetic pathways of actinorhodin and polycyclic tetramate macrolactams. Moreover, this streamlined genome is anticipated to facilitate directing the metabolic flux towards the production of desired compounds and increasing their yields.
PubMed: 38774831
DOI: 10.1016/j.synbio.2024.04.017 -
The ISME Journal Jan 2024Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut...
Host-microbe interactions underlie the development and fitness of many macroorganisms, including bees. Whereas many social bees benefit from vertically transmitted gut bacteria, current data suggests that solitary bees, which comprise the vast majority of species diversity within bees, lack a highly specialized gut microbiome. Here, we examine the composition and abundance of bacteria and fungi throughout the complete life cycle of the ground-nesting solitary bee Anthophora bomboides standfordiana. In contrast to expectations, immature bee stages maintain a distinct core microbiome consisting of Actinobacterial genera (Streptomyces, Nocardiodes) and the fungus Moniliella spathulata. Dormant (diapausing) larval bees hosted the most abundant and distinctive bacteria and fungi, attaining 33 and 52 times their initial copy number, respectively. We tested two adaptive hypotheses regarding microbial functions for diapausing bees. First, using isolated bacteria and fungi, we found that Streptomyces from brood cells inhibited the growth of multiple pathogenic filamentous fungi, suggesting a role in pathogen protection during overwintering, when bees face high pathogen pressure. Second, sugar alcohol composition changed in tandem with major changes in fungal abundance, suggesting links with bee cold tolerance or overwintering biology. We find that A. bomboides hosts a conserved core microbiome that may provide key fitness advantages through larval development and diapause, which raises the question of how this microbiome is maintained and faithfully transmitted between generations. Our results suggest that focus on microbiomes of mature or active insect developmental stages may overlook stage-specific symbionts and microbial fitness contributions during host dormancy.
Topics: Animals; Bees; Symbiosis; Bacteria; Fungi; Larva; Gastrointestinal Microbiome; Seasons; Host Microbial Interactions; Diapause
PubMed: 38767866
DOI: 10.1093/ismejo/wrae089 -
Chemical & Pharmaceutical Bulletin 2024Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the...
Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.
Topics: Multigene Family; Polyketides; Streptomyces; Molecular Structure; Polyketide Synthases; Molecular Conformation
PubMed: 38749722
DOI: 10.1248/cpb.c24-00144 -
BioRxiv : the Preprint Server For... May 2024In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal...
In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and β-farnesene.
PubMed: 38746203
DOI: 10.1101/2024.05.04.592530 -
Access Microbiology 2024sp. DSM 41014, DSM 41527, and DSM 41981 are three strains from the DSMZ strain collection. Here, we present the draft genome sequences of DSM 41014, DSM 41527, and DSM...
Genome sequence of the bialaphos producer Streptomyces sp. DSM 41527 and two putative phosphonate antibiotic producers Streptomyces sp. DSM 41014 and DSM 41981 from the DSMZ strain collection.
sp. DSM 41014, DSM 41527, and DSM 41981 are three strains from the DSMZ strain collection. Here, we present the draft genome sequences of DSM 41014, DSM 41527, and DSM 41981 with a size of 9.09 Mb, 8.45 Mb, and 9.23 Mb, respectively.
PubMed: 38737806
DOI: 10.1099/acmi.0.000770.v3 -
Current Research in Food Science 2024Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl...
Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl transfer, and crosslinking reactions between Ƴ-carboxamide groups of peptides or protein-bound glutamine and the Ɛ-amino group of lysine. In this study, we demonstrated an efficient systematic engineering strategy to enhance the synthesis of TGase in a recombinant smL2020 strain in a 1000-L fermentor. Briefly, the enzymatic properties of the TGase TG from . smL2020 and TGase TG from . smLD were compared to obtain the TGase TG with perfected characteristics for heterologous expression in a recombinant . smL2020ΔTG without the gene . Through multiple engineering strategies, including promoter engineering, optimizing the signal peptides and recombination sites, and increasing copies of the expression cassettes, the final TG activity in the recombinant smL2020ΔTG: (P--- ( BT1) reached 56.43 U/mL and 63.18 U/mL in shake flask and 1000-L fermentor, respectively, which was the highest reported to date. With the improvement of expression level, the application scope of TG in the food industry will continue to expand. Moreover, the genetic stability of the recombinant strain maintained at more than 20 generations. These findings proved the feasibility of multiple systematic engineering strategies in synthetic biology and provided an emerging solution to improve biosynthesis of industrial enzymes.
PubMed: 38736907
DOI: 10.1016/j.crfs.2024.100756 -
International Journal of Molecular... Apr 2024In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalNeoLect, from the first cloned wild-type...
In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalNeoLect, from the first cloned wild-type galactofuranosidase ( sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Gal, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalNGP), carrying the multivalent Gal units, as a suitable ligand and high-avidity system for the recognition of GalNeoLect which we successfully tested directly with the galactomannan spores of (ATCC 16404). Altogether, our results indicate that GalNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.
Topics: Galactose; Aspergillus; Lectins; Glycoproteins; Mannans; Animals; Serum Albumin, Bovine
PubMed: 38732045
DOI: 10.3390/ijms25094826